Synergistic Effects of Efflux Pump Modulators on the Azole Antifungal Susceptibility of Microsporum canis

The microbiologic and clinical resistance of dermatophytes is seldom reported, and the mechanisms associated with resistance are not well known. This study investigated the effect of efflux pump modulators (EPMs) (i.e., haloperidol HAL and promethazine PTZ) and their inhibiting activity on the minimum inhibitory concentrations of itraconazole (ITZ) and fluconazole (FLZ) against selected M. canis strains. M. canis strains with low (≤?1 μg/ml itraconazole and?<?64 μg/ml fluconazole) and high (>?1 μg/ml itraconazole and?≥?64 μg/ml fluconazole) azole MIC values were tested using Checkerboard microdilution assay. The disk diffusion assay, the minimum fungicidal concentration and the time-kill assay were also performed in order to confirm the results of checkerboard microdilution assay. The MIC values of ITZ and FLZ of M. canis decreased in the presence of subinhibitory concentrations of HAL and PTZ, the latter being more effective with a greater increased susceptibility. Synergism was observed in all strains with high azole MICs (FICI?<?0.5) and no synergism in the strains with low azole MICs. A fungicidal activity was observed after 48 h of incubation when ITZ and FLZ were tested in combination with HAL or PTZ. These results suggest that the drug efflux pumps are involved in the defense mechanisms to azole drugs in M. canis strains. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Complementary studies on M. canis resistance are advocated in order to investigate the molecular mechanisms of this phenomenon.

     

Bacterial L-arabinose isomerases: industrial application for D-tagatose production

D-tagatose is a natural monosaccharide with a low caloric value and has an anti-hyperglycemiant effect. This hexose has potential applications both in pharmaceutical and agro-food industries. However, the use of D-tagatose remains limited by its production cost. Many production procedures including chemical and biological processes were developed and patented. The most profitable production way is based on the use of L-arabinose isomerase which allows the manufacture of D-tagatose with an attractive rate. Future developments are focused on the generation of L-arabinose isomerases having biochemical properties satisfying the industrial applications. This report provides a brief review of the most recent patents that have been published relating to this area.     

Wild Boar (Sus scrofa) as Reservoir of Zoonotic Yeasts: Bioindicator of Environmental Quality

Mycopathologia - Wildlife animals are recognized as reservoirs for zoonotic fungi and their faeces might play an important role in introducing pathogens into the environment. Thought wild boar (Sus...     

The attractive recombinant phytase from Bacillus licheniformis: biochemical and molecular characterization

The phyL gene encoding phytase from the industrial strain Bacillus licheniformis ATCC 14580 (PhyL) was cloned, sequenced, and overexpressed in Escherichia coli. Biochemical characterization demonstrated that the recombinant enzyme has an apparent molecular weight of nearly 42 kDa. Interestingly, this enzyme was optimally active at 70–75 °C and pH 6.5–7.0. This enzyme is distinguishable by the fact that it preserved more than 40 % of its activity at wide range of temperatures from 4 to 85 °C. This new phytase displayed also a high specific activity of 316 U/mg. For its maximal activity and thermostability, this biocatalyst required only 0.6 mM of Ca²⁺ ion and exhibited high catalytic efficiency of 8.3 s^?1 μM^?1 towards phytic acid.     

The acid tolerant and cold-active β-galactosidase from Lactococcus lactis strain is an attractive biocatalyst for lactose hydrolysis

The gene encoding the β-galactosidase from the dairy Lactococcus lactis IL1403 strain was cloned, sequenced and overexpressed in Escherichia coli. The purified enzyme has a tetrameric arrangement composed of four identical 120 kDa subunits. Biochemical characterization showed that it is optimally active within a wide range of temperatures from 15 to 55 °C and of pH from 6.0 to 7.5. For its maximal activity this enzyme requires only 0.8 mM Fe²⁺ and 1.6 mM Mg²⁺. Purified protein displayed a high catalytic efficiency of 102 s^?1 mM^?1 for lactose. The enzyme stability was increased by immobilization mainly at low pH (from 4.0 to 5.5) and high temperatures (55 and 60 °C). The bioconversion of lactose using the L. lactis β-galactosidase allows the production of lactose with a high bioconversion rate (98 %) within a wide range of pH and temperature.     

Update of the spectrum of GJB2 gene mutations in Tunisian families with autosomal recessive nonsyndromic hearing loss

Hearing loss is the most frequent sensory disorder. It affects 3 in 1000 newborns. It is genetically heterogeneous with 60 causally-related genes identified to date. Mutations in GJB2 gene account for half of all cases of non-syndromic deafness. The aim of this study was to determine the relative frequency of GJB2 allele variants in Tunisia. In this study, we screened 138 patients with congenital hearing loss belonging to 131 families originating from different parts of Tunisia for mutations in GJB2 gene. GJB2 mutations were found in 39% of families (51/131). The most common mutation was c.35delG accounting for 35% of all cases (46/131). The second most frequent mutation was p.E47X present in 3.8% of families. Four identified mutations in our cohort have not been reported in Tunisia; p.V37I, c.235delC, p.G130A and the splice site mutation IVS1+1G>A (0.76%). These previously described mutations were detected only in families originating from Northern and not from other geographical regions in Tunisia. In conclusion we have confirmed the high frequency of c.35delG in Tunisia which represents 85.4% of all GJB2 mutant alleles. We have also extended the mutational spectrum of GJB2 gene in Tunisia and revealed a more pronounced allelic heterogeneity in the North compared to the rest of the country.     

Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji

Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l⁻¹) and BA (0.10–0.50 mg l⁻¹), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached 33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus). Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%) and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds.     

Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency

We have described previously the potential use of an alkaline protease from Bacillus pumilus CBS as an effective additive in laundry detergent formulations [B. Jaouadi, S. Ellouz-Chaabouni, M. Ben Ali, E. Ben Messaoud, B. Naili, A. Dhouib, S. Bejar, A novel alkaline protease from Bacillus pumilus CBS having a high compatibility with laundry detergent and a high feather-degrading activity, Process Biochem, submitted for publication]. Here, we purified this enzyme (named SAPB) and we cloned, sequenced and over-expressed the corresponding gene. The enzyme was purified to homogeneity using salt precipitation and gel filtration HPLC. The pure protease was found to be monomeric protein with a molecular mass of 34598.19Da as determined by MALDI-TOF mass spectrometry. The NH(2)-terminal sequence of first 21 amino acids (aa) of the purified SAPB was AQTVPYGIPQIKAPAVHAQGY and was completely identical to proteases from other Bacillus pumilus species. This protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine proteases superfamily. Interestingly, the optimum pH is 10.6 while the optimum temperature was determined to be 65 degrees C. The enzyme was completely stable within a wide range of pH (7.0-10.6) and temperature (30-55 degrees C). One of the distinguishing properties is its catalytic efficiency (k(cat)/K(m)) calculated to be 45,265min(-1)mM(-1) and 147,000min(-1)mM(-1) using casein and AAPF as substrates, respectively, which is higher than that of Subtilisin Carlsberg, Subtilisin BPN' and Subtilisin 309 determined under the same conditions. In addition, SAPB showed remarkable stability, for 24h at 40 degrees C, in the presence of 5% Tween-80, 1% SDS, 15% urea and 10% H(2)O(2), which comprise the common bleach-based detergent formulation. The sapB gene encoding SAPB was cloned, sequenced and over-expressed in Escherichia coli. The purified recombinant enzyme (rSAPB) has the same physicochemical and kinetic properties as the native one. SapB gene had an ORF of 1149bp encoding a protein of 383 aa organized into a signal peptide (29 aa), a pro-protein (79 aa) and a mature enzyme (275 aa). The deduced amino acid sequence inspection displays an important homology with other bacterial proteases. The highest homology of 98.1% was found with BPP-A protease from Bacillus pumilus MS-1, with only 8 aa of difference.     

Probing the essential catalytic residues and substrate affinity in the thermoactive Bacillus stearothermophilus US100 L-arabinose isomerase by site-directed mutagenesis

The L-arabinose isomerase (L-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 L-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 L-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the L-fucose isomerase and the L-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 L-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 L-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for L-fucose and decreased the affinity for L-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.     

Comparison of disc diffusion, Etest and agar dilution for susceptibility testing of colistin against Enterobacteriaceae

Aims: In this study, we compared different methods of colistin susceptibility testing, disc diffusion, agar dilution and Etest using a set of Enterobacteriaceae isolates that included colistin‐resistant strains. Methods and results: Susceptibility of 200 clinical isolates of Enterobacteriaceae to colistin was tested to compare agar dilution (reference method), disc diffusion (50 and 10 μg) and Etest. MICs (minimum inhibitory concentrations) were interpreted using the criteria established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Colistin exhibited excellent activity against Escherichia coli and E. cloacae (MIC₉₀ = 0·5 mg l^?1). In contrast, colistin was less active against Klebsiella pneumoniae (MIC₉₀ = 16 mg l^?1). Resistance rates varied from 0% in E. coli to 1·8% in E. cloacae and 13% in K. pneumoniae. High rates of very major errors were observed in the disc diffusion test using either the criteria of the Comité de l’antibiogramme de la Société Française de Microbiologie (CA‐SFM) or the criteria of the Clinical and Laboratory Standards Institute (CLSI), respectively, 3·5 and 2·5%. When the criteria of Gales et al. were applied, the number of very major errors was reduced to one (0·5%). The Etest showed good concordance with agar dilution method. Conclusion: Disc susceptibility testing methods are unreliable on detecting colistin resistance. MIC should be determined to confirm the susceptibility results by disc diffusion. Significance and Impact of the study: We recommend the determination of MIC by Etest for all multidrug‐resistant Enterobacteriaceae when colistin is required for the treatment.